Detection of filamentous and nitrifying bacteria in activated sludge with 16S rRNA probes

Activated sludge samples from wastewater treatment plants from potato starch and starch derivatives factory and from a municipal sewage treatment plant were analyzed with DNA probes specific for several filamentous bacteria. It was found that Haliscomenobacter hydrossis, Sphaerotilus natans, Thiothrix sp. and Eikelboom Type 021N were common in the activated sludges. Fluorescent in situ Hybridization (FISH) analysis could detect more types of sheathed bacteria and yielded a more accurate quantification of bacteria than conventional microscopy.In a pilot and a full scale wastewater treatment plant (WWTP) clear correlations were found between the SVI and growth of a Sphaerotilus natans and a Thiothrix sp. Addition of chlorine to the bulking sludge resulted in an improved SVI of the sludge but only damaged filamentous cells outside the floc. Nitrification was measured with substrate depletion and FISH analysis. Signal interpretation of FISH analysis was demonstrated both manually and with automated image analysis.     

Phylogenetic assessment of five filamentous bacteria isolated from bulking activated sludges

Nearly complete 16S ribosomal RNA (TRNA) sequences were determined for fully characterised axenic strains of Thiothrix, Eikelboom type O21N, and Eikelboom type 1701 originally isolated from bulking activated sludges. Thiothrix strains formed a monophyletic group (100% bootstrap support) with previously described Thiothrix nivea strain JP2 and Thiothrix ramosa. Eikelboom type O21N strain AP3 revealed a sufficiently strong relationship to the Thiothrix group to suggest a common ancestry for the two organisms although it was not possible to designate type O21N as a species of Thiothrix. Eikelboom type 1701 contained within its sequence the target sequence of an oligonucleotide probe for the detection of Sphaerotilus natans.     

In situ characterization of substrate uptake by Microthrix parvicella using microautoradiography

Microthrix parvicella is a filamentous microorganism responsible for bulking and foaming problems in many activated sludge treatment plants. The problems have increased with the introduction of nutrient removal in many countries, and presently, there is no reliable control strategy for M. parvicella. Little is known about the physiology of M. parvicella, and conflicting data exist about its preferred organic substrates, and whether it is able to be physiologically active under anaerobic and anoxic conditions. In this study, the ability of M. parvicella to take up various radioactively labeled organic compounds was investigated in situ at three nutrient removal plants using a microautoradiographic technique. Of 12 compounds tested under aerobic conditions only the long chain fatty acids (LCFA), oleic acid and palmitic acid, and to some extent a lipid, trioleic acid, were assimilated. None of the simple substrates such as acetate, propionate, butyrate, glucose, ethanol, glycine and leucine were taken up. Furthermore, the uptake of oleic acid was compared under anaerobic, anoxic and aerobic conditions, and it was demonstrated that in addition to aerobic conditions M. parvicella was also able to take up oleic acid under anaerobic and anoxic conditions. No difference in substrate uptake pattern for M. parvicella was found among the tested activated sludge plants. The results strongly indicate that a better control strategy against M. parvicella must rely on a better understanding of presence and availability of triglycerides and LCFA, and an improved knowledge of the physiology of M. parvicella under anaerobic and anoxic conditions.     

Effectiveness of oligonucleotide probes targeted against Thiothrix nivea and type 021n 16s rRNA for in situ identification and population monitoring in activated sludges

In situ hybridization (FISH) of two fluorescent oligonucleotide probes, TNI and 21N, directed against 16S rRNA fragments of Thiothrix nivea and type 021N filamentous bacteria, was used to study activated sludge samples of various origins. Comparison of the results obtained by this technique with morphological data enabled us to evaluate the sensitivity and specificity of the probes, as well as to propose practical classification criteria, in an effort to correlate the identification and ecology of these two microorganisms. We applied the two oligonucleotide probes to the study of Thiothrix sp. in pilot and laboratory batch reactors fed a substrate that was moderately deficient in rapidly available phosphorus. We monitored the growth dynamics of Thiothrix sp. in the pilot reactor, using various hydraulic configurations and after certain perturbations, such as transient substrate overloads. The results provided data which led us to question the importance of the length of time oxygen-deficient sludges spend in clarifier tanks and we discuss the effects of both moderate reductions in oxygen and of very intermittent feedings on the occurrence of Thiothrix sp. growth peaks. The identification and quantification of these filamentous bacteria by size class, made possible by in situ hybridization, are shown to be powerful tools for the early detection and evaluation of episodes of proliferation of filamentous bacteria, revealing them to be much more sensitive indicators than the sludge volume index (SVI).     

Occurrence and significance of filamentous bacteria in pulp and paper activated sludge systems.

A microbial survey of 27 activated sludge (AS) systems included 16 conventional activated sludge (CAS) systems, five sequential batch reactors (SBR) and six oxygen-activated sludge (OAS) systems, all treating pulp and paper effluents. The most prevalent filaments observed were Thiothrix (26%) and Type 021N (22%). The designs of the activated sludge systems seemed to have an effect on the filament types. We found Thiothrix to be the most common filament associated with bulking. For CAS systems, a completely mixed mode of operation promoted Thiothrix and Type 021N growth. Type 021N was favoured in CAS systems with food to microorganism (F/M) ratios higher than 0.2, and with dissolved oxygen (DO) residuals higher than 2 ppm, while Thiothrix generally proliferated at lower F/M ratio and DO residuals. Nutrient deficiencies as well as nutrient dosage variations were suspected in most of the systems having Thiothrix and Type 021N as the most prevalent filaments. Thiothrix appeared to prefer polyphosphate and/or ammonia rather than urea/phosphoric acid as N and P sources. Systems with aerobic selectors showed the lowest filament counts, while systems with no selectors showed the highest filament counts.     

Combining fluorescent in situ hybridization (fish) with cultivation and mathematical modeling to study population structure and function of ammonia-oxidizing bacteria in activated sludge

16S rRNA-targeted oligonucleotide probes for phylogenetically defined groups of autotrophic ammonia-oxidizing bacteria were used for analyzing the natural diversity of nitrifiers in an industrial sewage treatment plant receiving sewage with high ammonia concentrations. In this facility discontinuous aeration is used to allow for complete nitrification and denitrification. In situ hybridization revealed a yet undescribed diversity of ammonia oxidizers occurring in the plant. Surprisingly, the majority of the ammonia oxidizers were detected with probe combinations which indicate a close affiliation of these cells with Nitrosococcus mobilis. In addition, low numbers of ammonia-oxidizers related to the Nitrosomonas europaea - Nitrosomonas eutropha cluster were present. Interestingly, we also observed hybridization patterns which suggested the occurrence of a novel population of ammonia oxidizers. Confocal laser scanning microscopy revealed that all specifically stained ammonia oxidizers were clustered in microcolonies formed by rod-shaped bacteria. Combination of FISH and mathematical modeling was used to investigate diffusion limitation of ammonia and O₂ within these aggregates. Model simulations suggest that mass transfer limitations inside the clusters arc not as significant as the substrate limitations due to the activity of surrounding heterotrophic bacteria. To learn more about the ammonia-oxidizers of the industrial plant, we enriched and isolated ammonia-oxidizing bacteria from the activated sludge by combining classical cultivation techniques and FISH. Monitoring the isolates with the nested probe set allowed us to specifically identify those ammonia oxidizers which were found in situ to be numerically dominant. The phylogenetic relationship of these isolates determined by comparative 165 rDNA sequence analysts confirmed the affiliation suggested by FISH.     

Characterization of filamentous foaming in activated sludge systems using oligonucleotide hybridization probes and antibody probes

A quantitative method was developed for estimating Gordona mass in activated sludge foam and mixed liquor samples. The technique involves in situ hybridization with a genus-specific fluorescently labeled oligonucleotide probe calibrated on pure cultures of Gordona. The immunofluorescent technique of Hernandez et al. was modified to allow staining with fluorescently labeled antibody and hybridization probes. The results of this technique were compared to those from membrane hybridization studies using radioactively-labeled oligonuelcotide probes. Quantitative membrane hybridizations, in situ hybridizations, and antibody staining resulted in significantly different levels of Gordona in activated sludge foam, activated sludge mixed liquor, return activated sludge, and anaerobic digester sludge. Simultaneous staining with labeled antibodies and oligonucleolide probes provide a definitive identification for Gordona, and represents a new approach for in situ studies of this organism's role to foaming.     

Foaming in anaerobic digesters caused by Microthrix parvicella

Three large wastewater treatment plants in the greater Stockholm area have experienced serious anaerobic digester foaming. Microscopic studies of the sludge from the foam phase showed a network of the filamentous organism Microthrix parvicella. The morphology of the long, coiled filament appeared to be affected by the anaerobic conditions where it became broken up into to shorter and thicker filaments. The operating strategy to prevent foam in the anaerobic digesters at these plants is to control the growth of M. parvicella in the activated sludge tanks by increasing the sludge load. Top installed stirrers and the addition of poly-aluminium salt have also been used to prevent foam formation.     

A national survey of activated sludge separation problems in The Czech republic: Filaments,floc characteristics and activated sludge metabolic properties

The extent of activated sludge separation problems associated with excessive growth of filamentous microorganisms has increased recently in the Czech Republic. Most of the activated sludge plants in the country were contacted by mail with a questionnaire, the aim of which was to obtain the basic technological data on the plants and on the nature and intensity of activated sludge separation problems. Selected activated sludge plants were visited at different periods of the year and samples of activated sludge mixed liquor and foam were collected for microscopic examination and kinetic batch tests. It was found that good settling properties were associated more with good quality activated sludge flocs than with the absence of filaments. On the other hand, a very high abundance of filaments always resulted in bulking or foaming. The most common tilamentous microorganisms in activated sludge mixed liquors were M. parvicella, N. limicola and Types 0092,0041 and 0803 while the foams were mostly dominated by M. parvicella, NALOs (GALOs) and N. limicola. Seasonal shifts in filamentous population diversity were also observed. M. parvicella, Type 0092, N. limicola, Type 0803 and Type 0041 were dominant in both conventional and biological nutrient removal activated sludge plants whereas the other Eikelboom's types of filaments dominated mostly in conventional activated sludge plants. The ability of foams to denitrify depended on the dominating tilamentous microorganism and type of substrate.     

Evaluation of filamentous microorganism growth factors in an industrial wastewater activated sludge system

An investigation comprising four studies was undertaken to determine possible factors affecting the growth of several different types of filamentous microorganisms present in a bulking industrial wastewater activated sludge. Results from laboratory-scale continuous-flow and full-scale studies suggested that DO concentration and F:M ratio were the likely key factors affecting filamentous growth in the activated sludge. From the results of two laboratory studies isolating the effects of DO concentration and F:M ratio on filamentous growth, favorable growth ranges of DO concentration or F:M ratio were estimated for the following filaments: Microthrix parvicella, Nocardia spp., Nostocoida limicola II, and Types 0041, 1851, and 1863. Most of the bacteria causing filamentous bulking of the activated sludge were found to be filaments typically associated with low F:M, and increasing the F:M ratio appeared to cause N. limicola II to lose its competitive advantage in the activated sludge system. Type 1863, on the other hand, was found to be a low DO filament, as DO concentrations of 0.1 mg O₂/l or less appeared to be a necessary condition for its filamentous growth. Though Nocardia was found to be a low F:M filament, its growth also seemed to be affected by DO concentration, as its growth was stimulated by concentrations of 1.0 mg O₂/l or greater, with a near linear relationship up to at least 5 mg O₂/l.     

Impact of septic compounds and operational conditions on the microbiology of an activated sludge system.

In activated sludge (AS) biotreatment, septic compounds such as volatile organic acids and reduced sulphur compounds have been frequently cited as a major cause of Thiothrix and Type 021N filamentous bulking. These filaments are common in Canadian pulp and paper biotreatment systems, where they cause settling problems in secondary clarifiers. We conducted a 14-week study of a TMP/newsprint mill effluent to characterize the septic compounds entering the biotreatment, and to determine correlations with AS biomass characteristics and biotreatment operating parameters. A significant correlation was found between the sludge volume index, the abundance of Type 021N, and the propionic acid (PA) concentration in the primary clarified effluent. PA also induced a significant change in the flocculating bacteria size distribution determined by digital imaging. Consequently, the correlation observed between PA and Type 021N bulking is an indirect effect of inhibition of floc-forming microorganisms, giving a competitive advantage to filaments.     

Identification and quantification of Gordona amarae strains in activated sludge systems using comparative rRNA sequence analysis and phylogenetic hybridization probes

Small subunit (SSU) ribosomal RNA (rRNA) genes of four Gordona (Nocardia) amarae strains were sequenced and compared to the sequence of the G. amarae type strain obtained from the Ribosomal Database Project (RDP). Comparative sequence analysis showed that the five strains represent two lines of evolutionary descent: Group I consists of strains NM23 and ASACI and Group 2 contains strains SE-6. SE 102, and ASF3. To determine the abundance of G. amarae in activated sludge systems, we designed three oligonucleotide probes: a species-specific probe for G. amarae, a probe specific for Group I and a probe targeting Group 2. The probes were characterized by performing dissociation temperature and specificity studies. Using these probes, two other strains, strains SE-149B and RBI, also were found to be part of Group I. We used these probes along with previously designed probes in membrane hybridizations to determine the abundance of G. amarae, Group I, Group 2, Bacteria, Mycobacterium complex, and Gordona in samples from foaming episodes. We demonstrated that the Mycobacterium complex, the genus Gordona, and G. amarae strains were present in significantly greater concentrations in activated sludge foam than in mixed liquor.     

Identity, abundance and physiology of Aquaspirillum-related filamentous bacteria in activated sludge.

Microcolony-forming bacteria closely related to the genus Aquaspirillum in the Betaproteobacteria were recently observed to be abundant in many nutrient removal wastewater treatment plants. The developed oligonucleotide probe, Aqs997, however, occasionally also targeted some filamentous bacteria in activated sludge samples when fluorescence in situ hybridization was performed. In this study, the identity, abundance, and ecophysiology of these Aqs997-positive filamentous bacteria were studied in detail. Most of the Aqs997-positive filamentous bacteria could morphologically be identified as either Eikelboom Type 1701, Type 0041/0675 or possibly Type 1851, all characterized by epiphytic growth. They were found in almost all 21 wastewater treatment plants investigated. Two morphotypes were found. Type A filaments, which seemed to be the same genotype as the microcolony-forming bacteria targeted by probe Aqs997.Type B filaments also hybridized with probe GNS941, specific for the Chloroflexi phylum, so the true identity remains unclear. Aqs997-positive filaments usually stained Gram-negative, but Gram-positive filaments were also found, stressing the difficulties in identifying bacteria from morphology and simple staining results. Studies on the ecophysiology by microautoradiography showed that Aqs997-positive filamentous bacteria did not consume acetate and glucose, while some took up butyrate, mannose, and certain amino acids. Most likely, some Aqs997-positive filamentous bacteria were able to perform full denitrification such as the Aqs997-positive microcolony-forming bacteria, and some were able to store polyhydroxyalkanoates under anaerobic conditions, potentially being glycogen accumulating organisms.     

Occurrence and control of filamentous bulking in aerated wastewater treatment plants of the French paper industry.

The occurrence of filamentous bacteria was investigated in 15 French pulp and paper activated sludge wastewater treatment plant (WWTP). Large filamentous populations were present in most of the plants. Identification carried out with conventional methods based on morphological features and staining techniques showed that the four main filamentous bacteria encountered in these industrial WWTP and responsible for bulking belong to the genera Thiothrix sp., Type 021 N, Haliscomenobacter hydrossis and Type 0092. During two years a specific survey was performed for three of these WWTP showing recurrent bulking phenomena. Data from WWTP performance, chemical data and filaments characterization were compared to correlate the presence of specific filaments with process operating conditions.     

In situ structure/function studies in wastewater treatment systems

As an example of the importance and the potential of in situ structure/function studies, nitrifying aggregates from different zones of a lab-scale fluidised bed reactor were analysed by microelectrode measurements of O₂ NH₄⁺, NO₂⁻, and NO₃⁻ and in situ hybridisation targeting the 16S rRNA of the nitrifying bacteria. A shift trom an ammonia oxidising to a nitrite oxidising community is present along the reactor. In the single aggregates an active nitrifying shell of about 100 gm could be related to the maximum abundance of nitrifiers in this zone. Interestingly, the main actors in this system are not representatives of the well-described genera Nitrosomonas and Nitrobacter but some other ammonia oxidisers from the beta subclass of Protcobacteria and a thus far unknown nitrite oxidising population.     

Survey of filamentous populations in nutrient removal plants in four European countries

A joint EU research project aimed at solving activated sludge bulking in nutrient removal plants was initiated in 1993. The project started with a survey of the size and composition of the filamentous population in nutrient removal plants in Denmark, Germany, Greece and the Netherlands.The results show that biological nutrient removal process conditions indeed favour filamentous microorganisms in their competition with floc forming organisms. An increase in the size of the filamentous population resulted in a deterioration of the settling properties of the biomass, except for plants with Bio-P removal conditions. It is assumed that in the latter case the dense clusters of Bio-P bacteria increase the weight of the flocs, and compensate for the effect of the larger number of filaments.Although exceptions frequently occur, the following sequence in decreasing filamentous organism population size was observed for the process conditions indicated:

• -completely mixed + simultaneous denitrification;
• -completely mixed + intermittent aeration/denitrification;
• -alternating anoxic/oxic process conditions, with an anaerobic tank for biological phosphate removal (Bio-Denipho);
• -alternating anoxic/oxic process conditions (Bio-Denitro);
• -predenitrification

The surveys provided little information about the effect of nutrient removal in plants with plug flow aeration basins. Simultaneous precipitation with aluminium salts nearly always resulted in a low number of filaments and a good settling sludge.The size of the filamentous organism population showed a seasonal pattern with a maximum in winter/early spring and a minimum during summer (in Greece: during autumn). This seasonal variation is primarily caused by the effect of the season on the population sizes of M. parvicella, N. limicola and Type 0092.M. parvicella is by far the most important filamentous species in nutrient removal plants. In Denmark only, Type 0041 also frequently dominates the filamentous population, but seldom causes severe bulking. Considering their frequency of occurrence, approx. 10 other filamentous micro-organisms are of minor importance. Growth of some of these species, viz. those which use soluble substrate, can be prevented by the introduction of Bio-P process conditions.M. parvicella and Type 0041 (and probably also Actinomycetes and the Types 1851 and 0092) seem to compete for the same substrates i.e. the influent particulate fraction. Most of the differences in composition of the filamentous microorganism population can be explained by whether or not premixing of influent and recycled sludge is used. In general, premixing for a short period of time followed by anoxic conditions favours Type 0041. M. parvicella seems to proliferate if the particulate fraction is first hydrolysed or if it enters the plant via an oxic zone. It is concluded that bulking in nutrient removal plants is mainly caused by filamentous species requiring the particulate fraction for their growth.     

RNA/DNA ratio as an indicator of metabolic activity in resin acid-degrading bacteria

We investigated the relationship between the growth rate and the ratio of RNA to DNA in of four resin acid degrading bacteria isolated from a sequencing batch reactor (SBR). Chemical assays as well as slot blot hybndizations with species-specific oligonucleotide probes were used to quantify the nucleic acids. These slow-growing bacteria have a positive linear correlation between growth rate and RNA/DNA ratio, similar to faster-growing bacteria like E. coli. We propose to use this correlation to measure metabolic activities of selected resin acid degrading bacteria in the complex community of the SBR in situ using species-specific hybridization probes. Preliminary experiments suggest that hybridization probes can be used to detect growth rate-dependent changes in the RNA/DNA ratio.     

Substrate uptake by Gordonia amarae in activated sludge foams by FISH-MAR.

Gordonia amarae is a right-angled branching filament belonging to the mycolic acid-containing Actinobacteria which is commonly found in many foaming activated sludge wastewater treatment plants. Although studies on different substrates as sole carbon sources by pure cultures of G. amarae have been carried out, none have examined substrate uptake by this organism in situ. Uptake of several hydrophilic and hydrophobic substrates by G. amarae was evaluated in situ using a combination of fluorescence in situ hybridization and microautoradiography. G. amarae could assimilate a range of both hydrophilic and hydrophobic substrates. From the data, G. amarae appears to be physiologically active under aerobic, anaerobic and anoxic condition (NO2 and NO3) for some substrates. This might explain why attempts to control foaming caused by G. amarae using anoxic and anaerobic selectors have been unsuccessful. This study emphasizes that bacteria can behave differently in situ to pure cultures and that it is important to evaluate the in situ physiology of these bacteria if we are to better understand their role in the wastewater treatment process.     

In situ assessment of lampricide toxicity to age-0 lake sturgeon

The lampricides 3-trifluoromethyl-4-nitrophenol (TFM) and 2′, 5-dichloro-4′-nitrosalicylanilide (niclosamide) are used to control sea lamprey (Petromyzon marinus), an invasive species in the Great Lakes. Age-0 lake sturgeon (Acipenser fulvescens), a species of conservation concern, share similar stream habitats with larval sea lampreys and these streams can be targeted for lampricide applications on a 3- to 5-year cycle. Previous laboratory research found that lake sturgeon smaller than 100 mm could be susceptible to lampricide treatments. We conducted stream-side toxicity (bioassay) and in situ studies in conjunction with 10 lampricide applications in nine Great Lakes tributaries to determine whether sea lamprey treatments could result in in situ age-0 lake sturgeon mortality, and developed a logistic model to help predict lake sturgeon survival during future treatments. In the bioassays the observed concentrations where no lake sturgeon mortality occurred (no observable effect concentration, NOEC) were at or greater than the observed sea lamprey minimum lethal concentration (MLC or LC99) in 7 of 10 tests. We found that the mean in situ survival of age-0 lake sturgeon during 10 lampricide applications was 80%, with a range of 45–100% survival within streams. Modeling indicated that in age-0 lake sturgeon survival was negatively correlated with absolute TFM concentration and stream alkalinity, and positively correlated with stream pH and temperature. Overall survival was higher than expected based on previous research, and we expect that these data will help managers with decisions on the trade-offs between sea lamprey control and the effect on stream-specific populations of age-0 lake sturgeon.     

Molecular biological methods to detect “Microthrix parvicella” and to determine its abundance in activated sludge

Molecular biological methods were evaluated in attempts to detect and quantitate levels of “Microthrix parvicella” in activated sludges. Approximately 66% of the 23S rRNA gene sequence of a strain (Ben43) of the Gram positive bulking and foaming organism “Microthrix parvicella” was determined, while a lesser amount was determined for “M. parvicella” strain RNI. The high mo1%G+C Gram positive bacteria (HGCGPBs) possess two powerfully diagnostic regions in the 23S rDNA and these were investigated in both strains. Firstly, the 18 nucleotide HGCGPB probe sequence (HGC69a) varied in at least two nucleotides with the sequence from both strains of “M. parvicella”. Secondly, an approximately 100 nucleotide stable insert between helices 54 and 55 in the 23S rRNA of HGCGPBs was discovered to be present in “M. parvicella”, but in both strains it was unique in length (79 nucleotides) and sequence. The region of the 23S rDNA with the stable insert was exploited to develop a polymerase chain reaction assay in which amplicons from “M. parvicella” were larger than those from nonHGCGPBs (i.e. all Bacteria except the HGCGPBs), and smaller than those from HGCGPBs. This assay was evaluated with DNAs extracted from activated sludges but although “M. parvicella” was morphologically identified, and was a dominant filament in at least one of the samples, no “M. parvicella” specific sized amplicons could be recovered from it. Amplicons of sizes generated by nonHGCGPBs and HGCGPBs were routinely produced in the stable insert PCR with DNAs from activated sludges where the highest yield was of amplicons from nonHGCGPBS.A second series of experiments were undertaken with the objective of evaluating the use of a non-radioactive hybridization method, based on extraction of bacterial RNA, for quantifying “M. parvicella” in activated sludge samples. Total nucleic acids were extracted from activated sludge samples and immobilized on nylon membranes. Probing with 16S rRNA-directed DIG-labelled oligonucleotide probes, detection of chemiluminescent signals on membranes and densitometry allowed hybridization signals to be quantified. The relationship between the amount of nucleic acid hybridized and the hybridization signal intensity observed was found to be linear over a specified range of signal intensities. A range of activated sludge samples were analysed for “M. parvicella” and variations in levels could be distinguished.